Genomic Characterization of Salmonella enterica serovar Weltevreden Associated with Human Diarrhea

ABSTRACT Salmonella Weltevreden is an emerging pathogen associated with human diarrhea, and knowledge of the genomics and epidemiology of this serovar is still limited. In this study, we performed whole-genome sequencing of 96 S. Weltevreden isolates recovered from diarrheal patients and 62 isolates from food animals in China between 2006 and 2017. Together, with an additional 199 genome sequences of S. Weltevreden published in NCBI, we performed an analysis on all 357 S. Weltevreden genome sequences. Our results demonstrated that the majority of S. Weltevreden from diarrheal patients from China (97.92%, 94/96) and the other regions in the world (94.97%, 189/199) identified in this study were sequence type (ST) 365. The remaining types were ST3771 (n = 3), ST22 (n = 1), ST155 (n = 1), and ST684 (n = 1). In addition, ST365 was also widely recovered from animals, food, and environmental samples in different regions of the world. Phylogenetic analysis and pulsed-field gel electrophoresis (PFGE) revealed that S. Weltevreden from diarrheal patients was closely related to those recovered from food and environmental specimens. We also showed that S. Weltevreden did not exhibit severe antimicrobial resistance profiles, suggesting administering antibiotics is still effective for controlling the agent. Interestingly, we found that S. Weltevreden strains carried a number of virulence factor genes, and a 100.03-kb IncFII(S) type plasmid was widely distributed in S. Weltevreden strains. Elimination of this plasmid decreased the bacterial capacity to infect both Caco-2 cells and C57BL/6 mice, suggesting the importance of this plasmid for bacterial virulence. Our results contribute to the understanding of the epidemiology and virulence of S. Weltevreden. IMPORTANCE Salmonella Weltevreden is a pathogen associated with human diarrheal diseases found across the globe. However, knowledge of the genomics and epidemiology of this pathogen is still limited. In this study, we found S. Weltevreden sequence type (ST) 365 is commonly recovered from diarrheal patients in China and many other regions of the world, and there is no major difference between the Chinese isolates and the global isolates at the phylogenetic level. We also demonstrated that ST365 was widely recovered from animal, food, and environmental samples collected in different, global regions. Importantly, we discovered an IncFII(S) type plasmid commonly carried by S. Weltevreden strains of human, animal, and food origins, and this plasmid is likely to contribute to the bacterial pathogenesis. These findings enhance our understanding of the emergence of S. Weltevreden involved in diarrheal outbreaks and the global spread of S. Weltevreden strains.

updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.

Reviewer comments:
Reviewer #1 (Comments for the Author): The authors have addressed most of the concerns by the reviewers. However, some questions remain. A specific one is covered in lines 260-262, the authors only tried to conjugate the plasmids back into the parental strains, because that failed, they could try to simply transform the plasmids into the parental strains. The authors could consider doing this additional experiment.
We are grateful to your good comments and thank you for the useful suggestions. We have transformed the plasmid back in to the plasmid-curing strains through electroporation (lines 387-394), and have compared the invasion of plasmid-curing strains and plasmidcomplementary strains to Caco-2 cells (lines 401, 405). The results demonstrated that plasmidcomplementary strains displayed a stronger capacity of invasion than the plasmid-curing strains (lines 191-194, figure 4F). This is a really wonderful suggestion, and thank you very much for that.

Reviewer #2 (Comments for the Author):
Zhang and colleagues  presented an investigation on an emerging Salmonella serovar, previous epidemiology investigations suggest a possible water-related source, however, very few studies were reported in China, and the newly provided genomic data in this study would be a value add to the global dataset. However, there are some concerns that should be improved in the revised version.
Thank you very much for your comments and suggestions. Below please find the places for revision in the revised manuscript responded by us point-by-point: Line 29. how do you define "a comprehensive analysis", did you use all the data available in the global picture? or you did the genomic analysis other than AMR and VFs. There are many genetic factors or virulence factors that can be analyzed while it is missing in this study. I would be very careful by using this word.
We isolated and stored 118 strains but only 96 ones were recovered from the stocks. This is why we only sequenced 96 strains. We have illustrated this reason here. Please check lines 103 and 104. Thank you.
Line 109, 141 genomes are obviously selected from the database, but how to select these genomes should be reasonably documented. I believe most of this data is not published, it is deposited there.
Line 116, there is also a reason that should be stated here.
For these two concerns, the reason is there are only 199 assembled genome sequences, including 141 ones from humans and 58 ones from non-human species, in GenBank at the time of this paper writing (assessed as Nov 8, 2021; reference 20). We have added this reason in the manuscript as suggested. Please check lines 108-111. Thank you.
Line 163, is Caco-2 a large intestine cell? Did you use the right control strain for this invasion study, which strain are you comparing with?

Caco-2 cells (ATCC HTB-37) are epithelial cells isolated from colon tissue derived from a 72-
year-old (https://www.atcc.org/products/htb-37). Thank you for pointing this out for us. We have corrected our statement here (line 164).

This control strain we used at here is the virulent Salmonella Typhimurium ST19 strain SL1344 (PMID: 12704158). This strain could lead to serious intestinal infections in both humans and animals. It exhibits particularly high levels of SPI1 expression, and it is one of the comparator strains used in previously published in vitro and in vivo studies (PMID: 26933058). Also, we have added the comparator strain at this place. Please check line 166.
Thank you very much.
Line 170, there are a variety of plasmids detected here, how the logical rational link to one plasmid type, I would conduct a pan-genome analysis here to further understand the genetic diversity of this serovar.
We focus on this plasmid because it is distributed in 95.04% (268/282) of the plasmid-carrying S. Weltevreden strains analyzed in this study (line 173-174). It is known that plasmids in members of Enterobacteriaceae play important roles in bacterial antimicrobial resistance and virulence because many antimicrobial resistance genes and virulence factor genes are disseminated by plasmids (e.g., PMID: 33298160; PMID: 33172969). We therefore focus on the analysis of this plasmid. Thank you.
Thank you for your comments. We have sent emails to NCBI for requesting the release of the remaining genome sequences.

Line 368, what are the detection limits for your PCR method? and what is the copy number for this IncFII(S) type plasmid?
We verified the elimination of the plasmid using two PCR methods. Using the ordinary PCR method there is no band for the target gene and using the qPCR, there is no Ct value for the detection. However, we think the detection results did not affect the results because after the plasmid curing from the bacteria, their capacity of invading Caco-cells and mice decreased; however, after the plasmid being transferred back into the plasmid-curing strains, an increased capacity of invading to Caco-2 cells was observed compared to the plasmid-curing bacteria. Please see the results in lines 189-197, and figure 4. Thank you very much.
Line 418, did you use only one type of statistical analysis, I think several different experiments were used, and the mice infection experiment should be different.
Only "Multiple t tests" was used to compared data from different groups. After read your comments we have tried different methods on GraphPad Prism 8.0 and the results are similar. By searching published articles, we found statistical analysis based on "Multiple t tests" is acceptable (e.g., PMID: 36434990; PMID: 34686117; PMID: 34210738; PMID: 34408875; etc.). Thank you.

References, in general, are very old and should be updated with relevant and recent (within 5 years) work in the field.
Thank you for pointing this out for us. As we stated in the introduction section, Salmonella enterica serovar Weltevreden is a recently emerged pathogen. Therefore, there are not so many published articles. We have searched the key word "Salmonella enterica serovar Weltevreden" in PubMed and only 77 publications are obtained as of Nov 29, 2022, and only 23 of them are published within the recent five years. Notably, not all these 23 articles are related to the topic mentioned in our manuscript. On the contrary, many articles associated with the topics are published five years ago. This is why many citations are not very recent. Thank you.
Finally, the languages should be checked and improved by English-speaking experts, for readability and logical flow of the study.
Thank you for the suggestion. We have revised the manuscript carefully. Actually, before submission this manuscript has been edited by our two authors from Australia carefully, both of them are native speak with relevant academic background.
Some new references regarding Salmonella and Salmonellosis should be added. A big picture regarding the prevalence and disease burden in China should be the ideal contextual information for this study, even though this server is rarely studied in China. In other words, a more recent and comprehensive literature summary is needed in the introduction and discussion.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website. Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. There has been a large improvement in the readability of the manuscript and the inclusion of the reviewer recommended material has accomplished exactly what was hoped for. However, there are still many grammatical mistakes throughout the text, more than I feel comfortable accepting. Please resend the manuscript to your collaborators if needed as they missed many errors I found that include the following: Line 111-112: rephrase as "...from diarrheal stool and blood..." Line 119: "determined as" should be "determined to be" The sentence on lines 158-163 needs to be reworked. Line 167and 168: "resistant" should be "resistance" Line 172: "and these VFGs were" Line 176 "of" should be "for" Line 178 "where as" should be "whereas" Line 205 "to" should be "of" Line 206: "of" should be "with" Line 207: "more" should be "greater" Please modify the manuscript along the lines I have recommended. As these revisions are quite minor, I expect that you should be able to turn in the revised paper in less than 30 days, if not sooner. If your manuscript was reviewed, you will find the reviewers' comments below.
When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript. Detailed instructions on submitting your revised paper are below.

Link Not Available
Thank you for the privilege of reviewing your work. Below you will find instructions from the Microbiology Spectrum editorial office and comments generated during the review.
The ASM Journals program strives for constant improvement in our submission and publication process. Please tell us how we can improve your experience by taking this quick Author Survey. Sincerely,

Matthew Anderson
Editor, Microbiology Spectrum Reviewer comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Comments:
Thank you for submitting your manuscript to Microbiology Spectrum. As you will see your paper is very close to acceptance. There has been a large improvement in the readability of the manuscript and the inclusion of the reviewer recommended material has accomplished exactly what was hoped for. However, there are still many grammatical mistakes throughout the text, more than I feel comfortable accepting. Please resend the manuscript to your collaborators if needed as they missed many errors I found that include the following: The whole manuscript has been re-edited again by our two-coauthors from Australia (Dr Ziemanne and Dr Layton) carefully. All of them are native speaker with professional experience in Microbiology and Infectious Diseases. Those revised places are also marked in BLUE in the revised manuscript.